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. 2011 Jan 28;34(1):13-23.
doi: 10.1016/j.immuni.2010.12.017. Epub 2011 Jan 20.

Two-stage cooperative T cell receptor-peptide major histocompatibility complex-CD8 trimolecular interactions amplify antigen discrimination

Ning Jiang  1 , Jun Huang, Lindsay J Edwards, Baoyu Liu, Yan Zhang, Carrie D Beal, Brian D Evavold, Cheng Zhu
Affiliations

Two-stage cooperative T cell receptor-peptide major histocompatibility complex-CD8 trimolecular interactions amplify antigen discrimination

Ning Jiang et al. Immunity. .

Abstract

The T cell receptor (TCR) and CD8 bind peptide-major histocompatibility complex (pMHC) glycoproteins to initiate adaptive immune responses, yet the trimolecular binding kinetics at the T cell membrane is unknown. By using a micropipette adhesion frequency assay, we show that this kinetics has two stages. The first consists of TCR-dominant binding to agonist pMHC. This triggers a second stage consisting of a step increase in adhesion after a one second delay. The second-stage binding requires Src family kinase activity to initiate CD8 binding to the same pMHC engaged by the TCR. This induced trimeric-cooperative interaction enhances adhesion synergistically to favor potent ligands, which further amplifies discrimination. Our data reveal a TCR-CD8 positive-feedback loop involved in initial signaling steps that is sensitive to a single pMHC is rapid, reversible, synergistic, and peptide discriminative.

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Conflict of interest statement

We have no conflict of interest to declare.

Figures

Figure 1
Figure 1. Micropipette adhesion frequency assay
A, Micrograph of the micropipette assay. A T cell (left) was aspirated by a pipette and aligned with a pMHC-coated RBC held stationary by another pipette (right) (see Movie S1). B, Schematics of TCR and CD8 expressed on a T cell (left) and of pMHC coated on a RBC via biotin-streptavidin coupling (right). C, Specificity controls at contact duration of 0.25 s (solid bars) or 5 s (open bars) of adhesion frequencies between OT1 T cells and unmodified RBCs, biotinylated RBCs without coating, biotinylated RBCs coated with BSA, null pMHC-I (VSV:H-2Kb), pMHC-II (MOG:I-Ab) or agonist pMHC-I (OVA:H-2Kb), or between MOG CD4+ T cells and biotinylated RBCs coated with OVA:H-2Kb. Each T cell-RBC pair was tested repeatedly for 50 contact-retract cycles at a given contact duration to estimate an adhesion frequency, and 3-5 cell pairs were tested for each t to calculate a mean Pa ± s.e.m. See also Movie S1.
Figure 2
Figure 2. Two-stage kinetics of TCR-pMHC-CD8 trimolecular interaction
A, B, Pa vs. t T1 (A) or F5 (B) T cells interacting with RBCs bearing 25 OVA:H-2Kb/μm2 (A) or 14 NP68:H-2Db/μm2 (B) in the absence (■) or presence of anti-CD8 (○) or anti-TCR (△) reagents. Also included in A are data for OT1 T cells interacting with RBCs bearing 25 OVA:H-2Kbα3A2/μm2 (◇). Curves are trend lines. C, D, Pa vs. t data of OT1 (C) or F5 (D) T cells interacting with RBCs bearing 12 OVA:H-2Kb/μm2 or 14 NP68:H-2Db/μm2, respectively, at 25 (□) or 37 (○) °C. Representative data (measured by the same method as that in Fig. 1) of three repeated experiments are shown. See also Figure S1.
Figure 3
Figure 3. TCR-induced CD8-dependent increased adhesion can be differentially inhibited
Pa vs. t data (measured by the same method as that in Fig. 2) of OT1 (A, B) or F5 (C, D) T cells interacting with pMHC-coated RBCs in the absence (□) and presence (○) of PP2 (A, 3.5 OVA:H-2Kb/μm2), inhibitor for protein tyrosine phosphatase CD45 (B, 14 OVA:H-2Kb/μm2), Ca2+ chelator BAPTA-AM (C, 32 NP68:H-2Db/μm2), or MAP kinase kinase-1 inhibitor PD98059 (D, 32 NP68:H-2Db/μm2). DMSO treatment alone did not inhibit the second-stage adhesion increment. See also Figure S2.
Figure 4
Figure 4. Effect of anti-CD8 clone 53-6.7 on CD8 adhesion and its inhibition
Pa vs. t data (measured by the same method as that in Fig. 2) of OT1 T cells interacting with RBCs bearing indicated densities of OVA:H-2Kb (A, D), OVA:H-2Kbα3A2 (B), or GP33:H-2Db (C) in the absence (□) and presence (○, △) of the indicated mAb(s) or inhibitor. Note that the one-order-of-magnitude higher pMHC density used in panel C than in other panels translates to an order of magnitude lower binding (average number of bonds formed per pMHC density) between OT1 T cells and RBCs bearing H-2Db than H-2Kb (Methods, Eq. 3). See also Figure S3.
Figure 5
Figure 5. TCR engagement with a single pMHC is sufficient to induce cooperation with CD8
A, B, Pa vs. t data of OT1 (A) or F5 (B) T cells interacting with RBCs bearing indicated densities of OVA:H-2Kb or NP68:H-2Db. Curves are trend lines. C, Pa vs. t data of OT1 T cells interacting with RBCs bearing a mixture of OVA:H-2Kbα3A2 and VSV:H-2Kb at indicated ratios but the same total density (70 sites/μm2). Adhesion frequencies were measured by the same method as that in Fig. 2. See also Figure S4.
Figure 6
Figure 6. Second-stage binding does not need accumulation of TCR-CD8-pMHC trimolecular interaction and is reversible
A, For each contact duration, 50 pairs of F5 T cells and NP68:H-2Db-coated RBCs (32 μm−2) were each contacted once to estimate Pa from percent adherent pairs (□) to compare with mean Pa ± s.e.m. measured using 3-4 pairs of cells from the same batch each repeatedly contacted 50 times (○). B, F5 T cells and NP68:H-2Db-coated RBCs (32 μm−2) were repeatedly contacted 50 times first to measure Pa at a long duration (≥1 s, indicated by different symbols) and then another 50 times to measure anther Pa at a short duration (≤0.75 s, indicated by matched symbols) (□) to compare with Pa measured using cells from the same batch but repeatedly contacted 50 times at only one duration per pair (○). Data (mean ± s.e.m. of 3-4 cell pairs) at the first plateau overlap. C, Three pairs of F5 T cells and NP68:H-2Db-coated RBCs (23 μm−2) were each contacted 100 times with durations alternating between 0.25 and 5 s to measure two mean Pa ± s.e.m. (one at each contact duration) by dividing the number of adhesions resulting from contacts of the same duration by 50 (solid bars), which were significantly different (p = 0.0056, Student t-test). However, no statistical differences (p > 0.25) were found for both 0.25 and 5 s groups between these Pa values and those measured using 10 pairs of cells from the same batch each consecutively contacted 50 times (open bars). See also Movie S2.
Figure 7
Figure 7. Synergy between TCR and CD8 amplifies T cell discrimination
A–F, Pa vs. t data (measured by the same methods as that in Fig. 2) for OT1 T cells interacting with RBCs bearing H-2Kb (□) or H-2Kbα3A2 (○) complexed with OVA (A), A2 (B), G4 (C), E1 (D), V-OVA (E), and R4 (F). Also presented are data measured using RBCs bearing H-2Kb in the presence of the anti-TCR reagent B20.1 (△). Pa was converted to normalized adhesion bonds using Eq. 4 (Methods) and presented as mean ± s.e.m. Dashed curves represent trendlines to the sum of TCR-pMHC (○) and pMHC-CD8 (△) data. G, Steady-state (plateau values at 5 s) of the differential normalized adhesion bonds, △(<n>/mpMHC), formed between OT1 T cells and pMHC-coated RBCs minus the sum of those formed by the two bimolecular interactions for a panel of pMHC ligands with increasing potencies. H, The differential reciprocal concentrations required to reach half-maximal T cell proliferation △(1/EC50), measured in the absence of blocking antibody minus that measured in the presence of Fab of anti-CD8 mAb CT-CD8a, is plotted vs. △(<n>/mpMHC). See also Figure S5.
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